Hepatitis C virus (HCV), the major etiologic agent of transfusion acquired non-A, non-B hepatitis, is responsible for approximately 150,000 new cases of acute viral hepatitis annually in the United States. Approximately half of these infections progress to a chronic infection that can be associated with cirrhosis and/or hepatocellular carcinoma (Alter, et al., Science, 1992, 258, 135-140; and Alter, et al., New Eng. J. Med., 1992, 327, 1899-1905). In addition, HCV infection is an independent risk factor for the development of hepatocellular carcinoma as shown by the prevalence of anti-HCV antibodies (Colombo, et al., Lancet, 1989, ii, 1006-1008; Saito, et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 6547-6549; Simonetti, et al., An. Int. Med., 1992, 116, 97-102; and Tsukuma, et al., New Eng. J. Med., 1993, 328, 1797-1801).
HCV is an enveloped, positive stranded RNA virus, approximately 9,500 nucleotides in length, which has recently been classified as a separate genus within the Flavivirus family (Heinz, F. X., Arch. Virol. (Suppl.), 1992, 4, 163-171). Different isolates show considerable nucleotide sequence diversity leading to the subdivision of HCV genomes into at least eight genotypes (Simmonds, et al., J. Gen. Virol., 1993, 74, 2391-2399). In all genotypes, the viral genome contains a large open reading frame (ORF) that encodes a precursor polyprotein of 3010 to 3033 amino acids of approximately 330 Kd (Choo, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 2451-2455; Inchauspe, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 10292-10296; Kato, et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 9524-9528; Okamoto, et al., J. Gen. Virol., 1991, 72, 2697-2704; and Takamizawa, et al., J. Gen. Virol., 1991, 65, 1105-1113).
Individual HCV polypeptides are produced by proteolytic processing of the precursor polypeptide to produce core (C), envelope (E1, E2) and non-structural (NS2-NS5) proteins (Bartenschlager, et al., J. Gen. Virol., 1993, 67, 3835-3844; Grakoui, et al., J. Gen. Virol., 1993, 67, 2832-2843; and Selby, et al., J. Gen. Virol., 1993, 74, 1103-1113). This proteolysis is catalyzed by a combination of both cellular and viral encoded proteases.
In addition to the translated region, the HCV genome also contains both a 5' untranslated region (5' UTR) and a 3' untranslated region (3' UTR). The 5' UTR of 324 to 341 nucleotides represents the most highly conserved sequence among all HCV isolates reported to date (Han, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 1711-1715; and Bukh, et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 4942-4946). This 5' UTR has been postulated to contain important regulatory elements for replication and/or translation of HCV RNAs. The 5' UTR also contains several small open reading frames (ORF) but there is presently no evidence to suggest that these ORF sequences are actually translated.
The HCV core gene may be an important target for nucleic acid-based antiviral approaches. The first 191 amino acids of the HCV polyprotein precursor are believed to represent the viral nucleocapsid protein. This protein is comprised of a basic, RNA-binding amino-terminal domain and a highly hydrophobic carboxy-terminal region (Bukh, et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 8239-8243; and Santolini, et al., J. Virol., 1994, 68, 3631-3641). The mature 21 kDa core protein is cleaved from the polyprotein precursor by cellular signal peptidase and there is evidence to suggest that the HCV nucleocapsid protein is stably associated with the cytoplasmic surface of the endoplasmic reticulum membrane (Hijikata, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 5547-5551; and Santolini, et al., J. Virol., 1994, 68, 3631-3641). In contrast to the envelope glycoproteins which include a hypervariable region in the amino-terminal region of E2 (Weiner, et al., Virol., 1991, 180, 842-848; and Weiner, et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 3468-3472), the core protein is well conserved among the different HCV genotypes and generates a host immune response (Bukh, et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 8239-8243; and Houghton, et al., Hepatology, 1991, 14, 381-388). Previous studies have shown that the majority of HCV-infected individuals develop antibodies to the HCV core protein early in the course of infection (Chiba, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 4641-4645; Hosein, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 3647-3651; Hsu, et al., Hepatology, 1993, 17, 763-771; Katayama, et al., Hepatology, 1992, 15, 391-394; Nasoff, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 5462-5466; and Okamoto, et al., Hepatology, 1992, 15, 180-186). Furthermore, the nucleocapsid protein represents an important target for the cellular immune response against HCV (Botarelli, et al., Gastroenterol., 1993, 104, 580-587; Koziel, et al., J. Virol., 1993, 67, 7522-7532; and Shirai, et al., J. Virol., 1993, 68, 3334-3342). Finally, there are recent observations to suggest that the HCV core protein may have certain gene regulatory functions as well (Shih, et al., J. Virol., 1993, 67, 5823-5832). The high mutational rate of the viral genome probably occurs during viral replication and through immune selection. This phenomenon may be related to the establishment of persistent viral infection and subsequent disease chronicity (Weiner, et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 3468-3472; Kato, et al., Biochem. Biophys. Res. Comm., 1992, 189, 119-127; and Alter, et al., New Eng. J. Med., 1992, 327, 1899-1905).
The cellular immune events involved in liver damage and viral clearance during HCV infection have only partially been defined. In an attempt to examine a potential pathogenic role of liver-infiltrating lymphocytes in patients with chronic HCV infection, Koziel, et al. examined the cytotoxic T lymphocyte (CTL) response of such cells and demonstrated an HLA class I-restricted CD8+ CTL response that was directed against both structural and non-structural regions of HCV polypeptides (Koziel, et al., J. Virol., 1993, 67, 7522-7532; and Koziel, et al., J. Immunol., 1992, 149, 3339-3344). Other investigators have also noted the existence of CTLs in peripheral blood mononuclear cell populations that recognize epitopes on core and the other viral related proteins during chronic HCV infection (Kita, et al., Hepatol., 1993, 18, 1039-1044; and Cerny, et al., Intl. Symp. Viral Hepatitis Liver Dis., 1993, 83 (abstr.)).
Botarelli, et al. (Botarelli, et al., Gastroenterol., 1993, 104, 580-587) and Ferrari, et al. (Ferrari, et al., Hepatol., 1994, 19, 286-295) found HLA class II-restricted CD4+ T cell-mediated proliferative responses to several recombinant proteins derived from different regions of HCV in patients with chronic HCV infection. It is noteworthy that there was a correlation between T cell responses to HCV core protein, and a clinically benign course of the liver disease as well as subsequent eradication of the virus. However, a similar study showed the proliferative response to HCV core protein did not predict a benign clinical course with respect to the severity of the liver disease (Schupper, et al., Hepatol., 1993, 18, 1055-1060). Thus, it is important to clarify the association between active cellular immunity and the clinical course of the viral infection with respect to the type of liver injury and clinical response of HCV infection to IFN therapy. In this regard, studies involving peripheral blood mononuclear cell (PBMC) responses to a recombinant GST-HCV core fusion protein were conducted, and involved evaluating the ability of such cells to produce IFN-.gamma.; correlations were made to different clinical outcomes of HCV infection. It was found that mononuclear cells from 24 (52%) of 46 patients with chronic liver disease responded to the core protein; asymptomatic HCV carriers demonstrated a lower response rate (15%, p&lt;0.05). More important, individuals who had received IFN-A treatment and went into clinical and virologic emission had a higher response rate (75%, p&lt;0.05) to HCV core protein compared to those with ongoing hepatitis who failed therapy (31%). Of 25 patients whose mononuclear cells responded to HCV core protein, 18 had a significant response to one or more peptides; 12 patients reacted to a peptide mixture containing hydrophilic sequences. The core peptide amino acid sequence 140-160 was recognized by 9 patients. Interestingly, 7 of 8 patients bearing HLA DR4 and w53 haplotypes recognized the peptide sequence 141-160. Thus, the mononuclear cell response appeared to be HLA DR restricted and the responding cells were identified as CD4+ T cells. This study demonstrates the presence of immunodominant T cell epitopes within the HCV core protein in association with HLA DR phenotypes in patients with HCV associated liver disease.
Hepatitis B virus (HBV) is a major human pathogen for which there is no effective therapy. It is estimated that more than 300 million people are chronically infected with this virus worldwide. Exposure to HBV may result in acute or chronic hepatitis, liver cirrhosis and the development of hepatocellular carcinoma. The clinical consequences of this serious infection are of particular concern in the developing world where HBV infection is one of the leading causes of mortality and is also a major cause of acute and chronic liver disease in the United States and Europe as well. HBV is the prototype member of the hepadnavirus family, a group of closely related viruses (Ganem, et al., Annu. Rev. Biochem., 1987, 56, 651-693) that including, among others, the duck hepatitis B virus (DHBV) and the woodchuck hepatitis virus (WHV). Experimentally infected ducks or woodchucks faithfully reproduce many of the features of human disease such as acute and chronic infection and, in the case of chronically infected woodchucks, the development of hepatocellular carcinoma (Schodel, et al., "The Biology of Avian Hepatitis B viruses", In Molecular Biology of the Hepatitis B Virus, 1991, Vol.3, CRC Press, Boca Raton, Fla., pp.53-80; Korba, et al., J. Virol., 1989, 63, 1360-1370; and Korba, et al., Hepatology, 1989, 9, 461-470). Although viral replicative intermediates have been found in other tissues, the liver is the target organ and hepatocyte injury is associated with persistent viral infection. HBV per se appears not to be a cytopathic virus. It is likely, therefore, that the host immune response produced against viral epitopes produces the liver injury and treatment strategies designed to reduce viral replication in the liver may have beneficial clinical effects.
The HBV genome encodes for 4 open reading frames (ORF) that includes: 1) the S gene encoding for the envelope protein with 2 in-frame pre-S1 and pre-S2 polypeptides; 2) the polymerase ORF encoding for a reverse transcriptase protein that is responsible for reverse transcription of a 3.6 kb pregenomic RNA into DNA; 3) the core gene encoding for a protein that is assembled to complete the viral nucleocapsid; and 4) the HBx ORF encodes for a protein of unknown function. The pol gene encompasses 800% of the genome and overlaps with the other three ORFs. The core gene is preceded by an in-frame sequence that encodes for a signal peptide and following proteolytic cleavage gives rise to an antigenically distinct protein called the HBeAg. The HBx protein was found not to be essential for the viral life cycle in vitro (Blum, et al., J. Virol., 1992, 66, 123-127), but it appears to be necessary for the establishment of productive infection in vivo (Chen, et al., J. Virol., 1993, 67, 1218-1226). HBx can function as a transcriptional transactivator on a variety of cellular and viral genes and suggests that is may contribute to HCC development (Schek, et al., "The Hepadnaviral X Protein", In Molecular Biology of the Hepatitis B Virus, 1991, Vol.9, CRC Press, Boca Raton, Fla., pp.181-192).
Direct injection of DNA into animals is a promising method for delivering specific antigens for immunization (Barry, et al., Bio Techniques, 1994, 16, 616-619; Davis, et al., Hum. Mol. Genet., 1993, 11, 1847-1851; Tang, et al., Nature, 1992, 356, 152-154; Wang, et al., J. Virol., 1993, 67, 3338-3344; and Wolff, et al., Science, 1990, 247, 1465-1468). This approach has been successfully used to generate protective immunity against influenza virus in mice and chickens, against bovine herpes virus 1 in mice and cattle and against rabies virus in mice (Cox, et al., J. Virol., 1993, 67, 5664-5667; Fynan, et al., DNA and Cell Biol., 1993, 12, 785-789; Ulmer, et al., Science, 1993, 259, 1745-1749; and Xiang, et al., Virol., 1994, 199, 132-140). In most cases, strong, yet highly variable, antibody and cytotoxic T-cell responses were associated with control of infection. Indeed, the potential to generate long-lasting memory CTLs without using a liver vector makes this approach particularly attractive compared with those involving killed-virus vaccines and generating a CTL response that not only protects against acute infection but also may have benefits in eradicating persistent viral infection (Wolff, et al., Science, 1990, 247, 1465-1468; Wolff, et al., Hum. Mol. Genet., 1992, 1, 363-369; Manthorpe, et al., Human Gene Therapy, 1993, 4, 419-431; Ulmer, et al., Science, 1993, 259, 1745-1749; Yankauckas, et al., DNA and Cell Biol., 1993, 12, 777-783; Montgomery, et al., DNA and Cell Biol., 1993, 12, 777-783; Fynan, et al., DNA and Cell Biol., 1993, 12, 785-789; Wang, et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 4156-4160; Wang, et al., DNA and Cell Biol., 1993, 12, 799-805; Xiang, et al., Virol., 1994, 199, 132-140; and Davis, et al., Hum. Mol. Genet., 1993, 11, 1847-1851) of which HCV and HBV are important human diseases of world wide significance.
Presently, there is no universal, highly effective therapy of chronic HBV and/or HCV infection. Development of a vaccine strategy for HBV and/or HCV is complicated not only by the significant heterogeneity among HBV and HCV isolates, but also by the mixture of heterogeneous genomes within an isolate (Martell, et al., J. Virol., 1992, 66, 3225). In addition, the virus contains a highly variable envelope region.
Vaccination and immunization generally refer to the introduction of a non-virulent agent against which an individual's immune system can initiate an immune response which will then be available to defend against challenge by a pathogen. The immune system identifies invading "foreign" compositions and agents primarily by identifying proteins and other large molecules which are not normally present in the individual. The foreign protein represents a target against which the immune response is made.
PCT patent application PCT/US90/01348 discloses sequence information of clones of the HCV genome, amino acid sequences of HCV viral proteins and methods of making and using such compositions including anti-HCV vaccines comprising HCV proteins and peptides derived therefrom.
U.S. Ser. No. 08/008,342 filed Jan. 26, 1993, U.S. Ser. No. 08/029,336 filed Mar. 11, 1993, U.S. Ser. No. 08/125,012 filed Sep. 21, 1993, PCT patent application Ser. No. PCT/US94/00899 filed Jan. 26, 1994, and U.S. Ser. No. 08/221,579 filed Apr. 1, 1994 each contains descriptions of genetic immunization protocols. Vaccines against HCV are disclosed in each.
U.S. Ser. No. 08/318,248 filed Oct. 5, 1994, which is incorporated herein by reference, discloses genetic constructs comprising nucleotide sequences encoding HCV core protein which are useful as a vaccine. The HCV core DNA-based vaccine expresses high levels of core antigen in vitro and induces a strong immune response in vivo.
There remains a need for vaccines useful to protect individuals against hepatitis B virus and/or hepatitis C virus infection. There remains a need for methods of protecting individuals against hepatitis B virus and/or hepatitis C virus infection.